One of the best-studied phenomena in cell biology is the adhesion of cells to form an organized epithelium, which is facilitated by adherens junctions and desmosomes. In the testis, morphological studies in the 1970s reported the presence of desmosome-gap junctions (also known as desmosome-like junctions) between adjacent Sertoli cells at the blood-testis barrier, as well as between Sertoli cells and germ cells. Unfortunately, since this time there has not been a single report addressing the biology of desmosome-gap junctions and their significance in spermatogenesis. This application aims to fill this gap by investigating desmosome-gap junction dynamics in the mammalian testis. A survey by RT-PCR revealed that Sertoli and germ cells express several desmosome target genes, including desmoglein-2, desmocollin-2, plakophilin and desmoplakin. Moreover, when Sertoli cells were cultured at high density in vitro, thereby forming a polarized epithelium with functional cell junctions reminiscent of the blood-testis barrier in vivo, desmosome-like structures were visible by electron microscopy. This coincided with an up-regulation in the levels of several desmosome proteins, illustrating that the testis is equipped with the necessary 'building blocks'to establish functional desmosome-like junctions in vivo. In this application, the P.I. has two aims. First, using a well-characterized Sertoli cell in vitro model, the P.I. will investigate crosstalk between desmosome-like and gap junctions. To address this aim, the P.I. will use crosslinking and coimmunoprecipitation, followed by immunofluorescence microscopy, to identify protein-protein interactions common to both desmosome-like and gap junctions. Finally, the function of gap junctions will be assessed in functional experiments when two key desmosomal cadherins, namely desmoglein-2 and desmocollin-2, are silenced in Sertoli cells in vitro by RNAi. Second, the P.I. will examine the role of desmosome-gap junctions in blood-testis barrier function. Specifically, the P.I. will knockdown desmoglein-2 and desmocollin-2 in Sertoli cells and assess the function of the blood-testis barrier by transepithelial electrical resistance (TER) measurements, immunofluorescence and electron microscopy. Preliminary results presented herein, which showed changes in JAM-A, ZO-1 and CAR when only desmoglein-2 was silenced in Sertoli cells in vitro, support this Specific Aim. Finally, the P.I. will investigate in part the mechanism underlying blood- testis barrier restructuring following desmoglein-2 and desmocoolin-2. The P.I. believes these results will be welcomed in the field because they would represent the first findings on the biology of desmosome-gap junctions in the testis since their initial morphological identification. PUBLIC HEALTH RELEVANCE: Adhesion between adjacent Sertoli cells in the seminiferous epithelium of the testis in mammals is needed for spermatogenesis and fertility. If Sertoli cell adhesion at the level of the blood-testis barrier is compromised, germ cells deplete prematurely from the epithelium, and infertility can result. The study of desmosome-gap junctions in the testis will yield new and important insights into the biology of spermatogenesis.